B cell receptor (BCR)-mediated antigen internalization is the first step in the pathway of antigen processing and presentation, a phenomenon which is essential for cognate B cell-T cell interactions, B cell activation and establishment of immunological memory. However, the cellular and molecular mechanisms underlying the internalization of antigen-BCR (Ag-BCR) complexes remain ill defined, so much so that even the identity of the endocytosis motifs within the cytoplasmic domains of the BCR remain essentially uncharacterized. Therefore, the overall goals of this proposal are to establish the mechanisms of Ag-BCR internalization under different conditions of BCR ligation and cross-linking, and determine how modulation of the pathway of Ag-BCR endocytosis impacts important events such BCR-mediated antigen processing and presentation. Based on published work from this and other laboratories, we hypothesize that Ag-BCR internalization occurs via two distinct endocytic gateways, clathrin coated pits and plasma membrane lipid rafts, and that the level of engagement of these two distinct gateways depends upon the level of BCR cross-linking and BCR signaling-induced changes in the activity of BCR endocytosis motifs. Moreover, we propose that modulation of the relative roles of each of these gateways in Ag-BCR internalization will affect the mechanism of antigen processing and presentation. To test this hypothesis we will perform EM analysis of native membranes to determine the level of Ag-BCR internalization via plasma membrane clathrin coated pits and lipid rafts under different conditions of BCR ligation (Aim 1), identify and characterize the endocytosis motif(s) within the cytoplasmic tail of the BCR and determine the impact of signaling-induced BCR phosphorylation on endocytosis motif activity (Aim 2), and establish the impact of the mechanism of internalization on Ag-BCR ubiquitination (a novel phenomenon recently reported by our laboratory) as well as the intracellular trafficking, processing and presentation of internalized Ag-BCR complexes (Aim 3). Successful completion of these studies will result in a better understanding of the cellular and molecular mechanisms of BCR-mediated antigen internalization, processing and presentation. Moreover, application of these findings will allow for the development of improved vaccines and approaches to control autoimmunity.